Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulantory until later in life, and have near normal life expectancies. DMD mutations in the dystrophin gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
Several possible treatments have been investigated over the last 20 years, including myoblast-transplantation, DNA-targeted gene therapy, and antisense-mediated exon skipping (van Deutekom and van Ommen, (2003), Nat. Rev. Genet., 4(10):774-83). Antisense-mediated exon skipping aims at transforming out-of-frame mutations present in DMD patients into in-frame BMD-like mutations that result in synthesis of an at least partially functional dystrophin, which will prolong the viability of the muscles (Aartsma-Rus and van Ommen, (2007), RNA, 13(10): 1609-24).
Exon skipping can be induced by antisense oligonucleotides (AON) directed against the splice donor or splice acceptor site of a splice junction that are involved in the enzymatic process of exon joining, or against exon-internal sequences. In general, splice donor and splice acceptor sites comprise conserved sequences and targeting these sequences has the inevitable risk of co-targeting splice sites of additional exons from DMD or other gene transcripts.
Exon 44 of the DMD gene consists of 148 base pairs. Therapeutic skipping of exon 44 would restore the correct reading frame in DMD patients having deletions including but not limited to exons 03-43, 05-43, 06-43, 10-43, 13-43, 14-43, 17-43, 19-43, 28-43, 30-43, 31-43, 33-43, 34-43, 35-43, 36-43, 37-43, 38-43, 40-43, 41-43, 42-43, 43, 45, 45-54, and 45-68, or having a duplication of exon 44. Furthermore, for some DMD patients the mutations are such that the simultaneous skipping of one or more exons is required in addition to exon 44 skipping to restore the reading frame. Non-limiting examples of such mutations are nonsense point mutations in the flanking exons 43 or 45, requiring exon 43+44 skipping or exon 44+45 skipping respectively. The aforementioned mutations in total occur in about 6-8% of all DMD patients. The majority of resulting dystrophin proteins will be truncated in the central rod domain of the protein, leaving the essential N-terminal actin-binding domain and the C-terminal domain binding to dystrobrevin and syntrophin, and the β-dystroglycan-binding C-terminal cysteine-rich domain, intact.